With the invention of the Gamma Camera, and, just as importantly, with the invention of better high-speed imaging machines, pharmaceutical substances with radioactive “tags” have become extremely important in medical imaging and treatment. The concept is that a compound, or just as often, a part of a compound, called a ligand, sometimes referred to as an “agent” or which bonds to some other substance, is designed to target a particular area of a mammal's body or a particular type of tissue or molecule in that body. The compound, ligand or agent will be referred to as a ligand for convenience sake. The mammal this is most often used on is the human body, and references in this invention to a human are equally applicable to any mammal, or for that matter to any animal or plant.
For instance, certain ligands tend to concentrate in heart muscle tissue. The concept behind radiopharmaceutical imaging is to “tag” that ligand with a radioactive substance, i.e. radioactively mark a substance to create an “imaging agent,” so that a health care provider can find out where the ligand exists or is concentrating. By administering the radioactively tagged ligand, and placing the patient in an imaging machine, a health care provider can “look inside” a patient's body to assist in therapy or diagnosis. If a person has poor heart circulation, the radionuclide tagged ligand, such as Tc 99m TIBI, will not be well-circulated to areas of the heart muscle which have compromised blood flow, enabling evaluation of a person's “heart condition.” Importantly, the health care provider can often “look inside” without having to actually cut open or invade the body (non-invasive technique), or can minimize bodily invasion. Obviously, the continued presence of radioactive substances is not desirable, so substances are selected with a short “half-life.” The half-life is a time defined as the time in which the radioactive emission declines by one-half. The diminution of radioactivity is referred to as radioactive decay. Between the body washing out the radiopharmaceutical substances used in conjunction with this invention, and the use of substances with a short half-life, the amount of a patient's radioactive exposure is minimized.
Radioactive pharmaceuticals are in common use in imaging studies to aid in the diagnosis of a wide variety of illnesses including cardiac, renal and neoplastic diseases. These pharmaceuticals, known in the art as “imaging agents,” typically are based on a gamma-emitting radionuclide attached to a carrier molecule or “ligand.” Gamma-emitting radionuclides are the radionuclides of choice for conducting diagnostic imaging studies because, while gamma emitting radiation is detectable with appropriate imaging equipment, it is substantially less-ionizing than beta or alpha radiation. Thus, gamma emitting radiation causes minimal damage to targeted or surrounding tissues.
Radioactive pharmaceuticals now are finding increased use as diagnostic agents for finding neoplastic disorders, especially tumors. Diagnostic radiopharmaceuticals generally incorporate a gamma emitting radionuclide, the radiation emission being useful in the detection of certain neoplastic disorders.
The radioactive marking or tagging is often done by complexing the radioactive substance inside a group of ligands, that is surrounding it by a complex of ligands, so that the desired chemical characteristics are expressed toward the exterior of the complex with the tag shielded by the outer complex and simply carried along as a marker. The entire complex with the radioactive element, also called a radionuclide, functions as a radioactive marker, and can be more generally referred to as a radiopharmaceutical.
The use of small quantities of drugs used for such activities is desirable for cost reasons, and it is desirable to minimize the amount of radioactive substance used.
While the efficacy of radioactive diagnostic and therapeutic agents is established, it is also well known that the emitted radiation can cause substantial chemical damage or destabilization to various components in radiopharmaceutical preparations, referred to as autoradiolysis. Emitted radiation causes the generation of free radicals in water solutions, which free radicals are generally peroxides and superoxides. Such free radicals can precipitate proteins present in the preparations, and can cause chemical damage to other substances present in the preparations. Free radicals are molecules with unbonded electrons that often result because the emissions from the radioactive element can damage molecules by knocking apart water molecules forming hydroxyl radicals and hydrogen radicals, leaving an element or compound with a shell of charged electrons which seek to bond with other molecules and atoms and destabilize or change those molecules and atoms. The degradation and destabilization of proteins and other components caused by the radiation is especially problematic in aqueous preparations. Under the present art, the radiolysis causes the aqueous stored ligand and radioactive isotope bonded to the ligand to degenerate and destroys the complex which renders it useless for imaging because the biological characteristics that localize the complex to a tissue are gone. The degradation or destabilization lowers or destroys the effectiveness of radiopharmaceutical preparations, and has posed a serious problem in the art. Wahl, et al, Journal of Nuclear Medicine, Vol 31, Issue 1 84-89, discuss the fact that freezing radiolabeled antibodies at −70 degrees C. stabilizes the molecule for an indefinite period but 80 to 90% of the immunoreactivity is lost in as little as 24 hours when stored at 4 degrees C.
If the ligands are permitted to reside with the radioactive elements for an extended period, particularly in an aqueous (water-based) solution, the radiolysis is increased. Thus, any process to reduce the compounds to dried form has to be rapid and yield predictable result. Further, to avoid the higher concentrations and protect the ligands, presently the radiopharmaceutical solution is diluted, but that in itself only slows the drying time and complicates the problem and increases the unpredictability of the non-radioisotope portion of the radiopharmaceutical because of radiolysis. Heating the radiopharmaceutical in solution to accelerate the drying and removal of water has the undesirable effect of potentially damaging the ligand since chemical activity normally increases upon heating or injection of energy and therefore the effects of radiolysis are also increased during this prolonged drying period with heating. Most proteins are badly damaged upon heating. Certain ligands, such as isonitrile, simply evaporate and disappear upon heating. Further, minimization of localized heating at an atomic scale is important to preserve both the small quantities needed and to yield a specific concentration of desired product.
Wolfangel, U.S. Pat. No. 5,219,556, Jun. 15, 1993, entitled stabilized therapeutic radiopharmaceutical complexes, expressed his concern as follows: “The isotopes which are most useable with this process are determined by practical considerations. Again, Tc-99m would be a poor candidate for use since its six-hour half-life makes lyophilization impractical, as the lyophilization step itself generally takes about 24 hours to perform.”
Facially, the '556 invention seemed to identify a useful process and resulting composition, but the lyophilization step in '556 invention, as the application stated, took about 24 hours. The '556 invention stated: “The lyophilization is carried out by pre-freezing the product, and then subjecting the frozen product to a high vacuum to effect essentially complete removal of water through the process of sublimation. The resultant pellet contains the complex in an anhydrous form which generally can be stored indefinitely, with practical consideration being given to the half-life of the radionuclide. The intended period of storage for radiopharmaceutical products is thus practically limited by the half-life of the radionuclides. In the case of Re-186, for example, the desired period of storage would range from 7 to about 30 days. Thus, this pellet can be shipped to the end users of the product and reconstituted with a diluent at the time of administration to the patient with very little effort on the part of the health care professional and/or nuclear pharmacist.”
Because the procedures in '556 did not rapidly lyophilize the product, and contemplated a 24 hour period for lyophilization, the claims of '556 invention were necessarily limited to utilization of a “therapeutic amount of an alpha- or beta-emitting radionuclide.” Wolfangel had observed that compounds with a half-life of at least 12 hours are preferred. By contrast, the use of Tc-99m, which also emits gamma rays, with a half-life of only six hours, or the use of other similarly short-lived radioisotopes, becomes impractical.
Wolfangel '556 proposed in his example 1 to first lyophilize certain compounds, add the radionuclide complex, sparge with gas, seal the vial and then heat it. Unfortunately, the heating to 100 degree C. renders the procedure useless in conjunction with most proteins or peptides, and many commonly used complexes. Further, the proposal was to use 1 ml of sodium perrhenate Re-186 containing 1 mg of rhenium, with water added to produce 3 ml. The quantities contemplated were substantial and exposed the workers to substantial amounts of radiation. In example 3, it was proposed that the complex be frozen to −30 degree C. or colder and then apply a vacuum, but it was proposed to apply shelf heat at 6 degree per hour until a product temperature of 30 degree C. was reached, at which time the temperature would be held for two hours. That would require 12 hours. The procedure suffered from the infirmity of not quickly removing water and therefore not preventing radiolysis of the water and not preventing the generation of free radicals which damage the complexes. The second example 2 followed the first, but used smaller quantities, and proposed heating. Example 3 proposed heating to 85 degree C. for 30 minutes which would destroy most proteins and thereafter freezing and lyophilizing the sealed vials.
For diagnostic imaging purposes, radiopharmaceuticals based on a coordination complex comprised of a gamma-emitting radionuclide and a chelate have been used to provide both negative and positive images of body organs, skeletal images and the like. The Tc-99m skeletal imaging agents are well-known examples of such complexes. One drawback to the use of these radioactive complexes is that while they are administered to the patient in the form of a solution, neither the complexes per se nor the solutions prepared from them are overly stable. Consequently, the coordination complex and solution to be administered commonly are prepared “on site,” that is, they are prepared by a nuclear pharmacist or health care technician just prior to conducting the study. The preparation of appropriate radiopharmaceutical compositions is complicated by the fact that several steps may be involved, during each of which the health care worker must be shielded from the radionuclide.
The preparation of stable radiopharmaceutical diagnostic agents, due to the type of radioactivity, presents even greater problems. These agents typically are based on a relatively energetic gamma emitting radionuclide complexed with a chelate. Frequently, the radionuclide/chelate complex is in turn bound to a carrier molecule which bears a site-specific receptor. Thus, it is known that a gamma emitting radionuclide attached to a tumor-specific antibody or antibody fragment can destroy targeted neoplastic or otherwise diseased cells via exposure to the emitted ionizing radiation. Bi-functional chelates useful for attaching a diagnostic radionuclide to a carrier molecule such as an antibody are known in the art. See e.g. Meares et al., Anal. Biochem. 142:68-78 (1984).
For most imaging and diagnostic applications of radiopharmaceutical complexes of the types mentioned above, the nonradioactive portion(s) of the complex is prepared and stored until time for administration to the patient, at which time the radioactive portion of the complex is added to form the radiopharmaceutical of interest. For example, attempts to prepare radionuclide-antibody complexes have resulted in complexes which must be administered to the patient just after preparation because, as a result of radiolysis, immunoreactivity may decrease considerably after addition of the radionuclide to the antibody. In Mather et al., J. Nucl. Med., 28:1034-1036 (1987), a technique for labeling monoclonal antibodies with large activities of radio iodine using the reagent N-bromosuccinimide is described. The authors suggest that the antibodies labeled in this manner be administered to the patient immediately after preparation to avoid losses of immunoreactivity. Other examples of the preparation of the nonradioactive portion of the complex followed by on-site addition of the radioactive portion are disclosed in U.S. Pat. No. 4,652,440 (1987). Further, in many situations, the radioactive component of the complex must be generated and/or purified at the time the radiopharmaceutical is prepared for administration to the patient. U.S. Pat. No. 4,778,672 (1988) describes, for example, a method for purifying pertechnetate and perrhenate for use in a radiopharmaceutical.
According to Wolfangel '556, EP 250,966 (1988) describes a method for obtaining a sterile, purified, complexed radioactive perrhenate from a mixture which includes, in addition to the ligand-complexed radioactive perrhenate, uncomplexed ligand, uncomplexed perrhenate, rhenium dioxide and various other compounds. Specifically, the application teaches a method for purifying a complex of rhenium-186 and 1-hydroxyethylidene diphosphonate (HEDP) chelate from a crude solution. Because of the instability of the complex, purification of the rhenium-HEDP complex by a low pressure or gravity flow chromatographic procedure is required. The purification procedure involves the aseptic collection of several fractions, followed by a determination of which fractions should be combined. After combining the appropriate fractions, the fractions are sterile-filtered and diluted prior to injection into the patient. The purified rhenium-HEDP complex should be injected into the patient within one hour of preparation to avoid the possibility of degradation. The rhenium complex may have to be purified twice before use, causing inconvenience and greater possibilities for radiation exposure to the health-care technician.
While the lyophilization process has been applied to various types of pharmaceutical preparations in the past, the notion of lyophilizing short lived gamma emitting radiopharmaceutical preparations has not been addressed. In part, this is believed to be due to skepticism of those skilled in the art that such a procedure could be safely carried out. U.S. Pat. No. 4,489,053 (Azuma et al.; Dec. 18, 1984) relates to Tc-99m-based diagnostic imaging agents. The patentee notes that the non-radioactive agents may be prepared in lyophilized form and that stabilizers are required to prevent radiolysis once the Tc-99m is added.
Thus, there is a need in the art for a method of centrally preparing and purifying a stabilized diagnostic radiopharmaceutical for shipment to the site of use in a form ready for simple reconstitution prior to its administration in diagnostic applications without the necessity of additional stabilizers. Because of the length of the Wolfangel process, many of the protein combinations with radionuclides are impractical because of the sensitivity of the protein in combination to any free radical attack caused by radioactive decay, and thus the present invention is a novel means to enable practical commercial use of radionuclide labelled proteins and peptides. The length also effectively prohibits the use of shorter half life radionuclides because in order to use them with the Wolfangel process, the concentrations of the radionuclides have to be increased to account for the several half lives during the 24 hours lyophilization and the time for shipment, which concentration exposes workers to higher concentrations of radioactivity and which time exposes the ligands to radiolysis which decreases their predictability of use in the patient, if they are effective at all. If, in order to avoid the higher concentrations, more dilute amounts are used, then the quantity of liquid involved jeopardizes the efficacy of lyophilization. There is a particular need in the art for a method of centrally preparing and purifying radionuclide-labeled antibodies and antibody fragments, owing to their relatively unstable immunoreactivities once in aqueous solution. Most particularly, this invention enables the use of short-half-life radionuclides with ligands potentially subject to radiolysis that are stable with useful shelf life at room temperatures that can be shipped in a commercially cheaper manner, and easily reconstituted.